Amide compounds as boosters of antivirals

ABSTRACT

The present invention relates to compounds of formula (I) that have CYP450 inhibiting properties and are useful as boosting agents when used with HIV treatments. These compounds are represented by formula 
                         
including the salts and stereoisomeric forms thereof, wherein R 1  is 5-thiazolyl or 3-pyridinyl; R 2  is iso-butyl, 2,2-dimethylpropyl; 2-hydroxy-2-methyl-propyl or cyclohexylmethyl; R 3  is phenyl optionally substituted with one or more halogens, trifluoromethyl, C 1-6 -alkyl or C 1-6 -alkoxy wherein optionally two of said alkoxy groups are be linked to each other to form a 5 or 6-membered ring; heteroaryl; C 3-7 cycloalkyl optionally substituted with one or more halogens; C 1-6 alkyl optionally substituted with heteroaryl; —O—CH 2 — (heteroaryl). As boosting agents they are able to increase at least one of the pharmacokinetic variables of certain drugs when co-administered, or improve the bioavailability of certain drugs. Methods for the preparation of the compounds of the invention and pharmaceutical compositions comprising these compounds are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national stage of PCT Application No.PCT/EP2009/062996, filed Oct. 7, 2009, which claims priority fromEuropean Patent Application No. 08166004.5, filed Oct. 7, 2008, all ofwhich are hereby incorporated by reference in their entirety.

The present invention relates to compounds that have CYP450 inhibitingproperties and are therefore useful as boosters of certain drugs, i.e.they are able to increase at least one of the pharmacokinetic variablesof certain drugs when co-administered. The invention further providesthe use of said compounds as improvers of the bioavailability of certaindrugs. Methods for the preparation of the compounds of the invention andpharmaceutical compositions comprising these compounds are alsoprovided.

Many drugs, including some HIV protease inhibitors (PIs) andnon-nucleoside reverse transcriptase inhibitors (NNRTIs), aremetabolized by the cytochrome P450 system. The cytochrome P450 system isa group of enzymes found in the liver and the gut, which have a numberof functions in the human body. The activity of cytochrome P450 differsbetween individuals and between populations. Small genetic variationscan affect how many particular enzymes are expressed, and thus howquickly the drug is metabolized.

Cytochrome P450 enzymes which derive from a particular gene are calledisoforms. Based on the similarity of their chemical make-up, isoformsare divided into families and subfamilies. Enzyme variants are describedthrough a numbering and lettering system, which reflects their chemicaland genetic structure.

Cytochrome P450, subfamily IIIA (niphedipine oxidase), polypeptide 4,also referred to as CYP3A4, is one particular metabolic pathway used forbreakdown and clearance of medications and other substances.

Metabolization of certain drugs by the cytochrome P450 system frequentlyresults in said drugs having unfavourable pharmacokinetics and the needfor more frequent and higher doses than are most desirable.Administration of such drugs with an agent that inhibits metabolism bythe cytochrome P450 system may improve the pharmacokinetics of the drug.In this respect, methods for improving the pharmacokinetics of certaindrugs have been published, see, e.g., U.S. Pat. No. 6,037,157; D. E.Kempf et al. Antimicrob. Agents Chemother., 41, pp. 654-660 (1997).

In WO03/049746 there is disclosed a method for improving thepharmacokinetics of hexahydrofuro[2,3-b]furanyl containing HIV proteaseinhibitors comprising administering to a human in need thereof acombination of a therapeutically effective amount of ahexahydrofuro[2,3-b]furanyl containing HIV protease inhibitor, and atherapeutically effective amount of a cytochrome P450 inhibitor.

Most HIV protease inhibitors in clinical therapy are now paired withritonavir to improve exposure and thereby enhancing clinical efficacy.This type of applied drug-drug interaction is referred to as “boosting”.Boosting also supports simplified treatment regimens for current PIs byreduction of pill burden and frequency of daily intakes.

Unfortunately, ritonavir enhancement of PI regimens, even at low doses,is not without risk. Ritonavir is itself an HIV protease inhibitor.Resistance to ritonavir is associated with the selection of one or moreof several resistance mutations. Resistance mutations selected byritonavir frequently confer or contribute to resistance against otherprotease inhibitors. Different mutations are associated withcross-resistance to different drugs. For example, M46I is associatedwith cross-resistance to indinavir, nelfinavir, and fosamprenavir (butnot to saquinavir); V82A,F,T,S alone is associated with cross-resistanceto indinavir, but in combination with other mutations also confersresistance to nelfinavir, fosamprenavir, and saquinavir; and I84Vcontributes to resistance against all available protease inhibitors.While no single one of these mutations is associated with fullresistance to lopinavir, each contributes partial resistance, and thepresence of several mutations together can confer resistance. Responseto indinavir is unlikely in the setting of resistance to ritonavir.

As such, there is a high medical need for alternatives to ritonavir asboosting agent in an effective and safe anti-HIV treatment. There isalso a high medical for alternatives to ritonavir as boosting agent inan effective and safe anti-HIV treatment wherein the possibility ofresistance development due to the boosting agent is excluded.

In accordance with the present invention it has now been found that thefollowing compounds of formula (I) have CYP450 inhibiting properties andare useful as boosting agents. These compounds are represented byformula

the salts and stereoisomeric forms thereof, whereinR₁ is 5-thiazolyl or 3-pyridinyl;R₂ is iso-butyl, 2,2-dimethylpropyl; 2-hydroxy-2-methyl-propyl orcyclohexyl-methyl;R₃ is phenyl optionally substituted with one or more halogens,trifluoromethyl, C₁₋₆-alkyl or C₁₋₆-alkoxy wherein optionally two ofsaid alkoxy groups are be linked to each other to form a 5 or 6-memberedring; heteroaryl; C₃₋₇cycloalkyl optionally substituted with one or morehalogens; C₁₋₆alkyl optionally substituted with heteroaryl; —O—CH₂—(heteroaryl).

Preferred compounds are those compounds wherein R₁ is 5-thiazolyl, R₂ isiso-butyl and R₃ is quinoxalyl.

Most preferred is the compound having the formula (II)

the salts and stereoisomeric forms thereof, said compound having thechemical name1S-2R-{1-Benzyl-2-hydroxy-3-[isobutyl-(quinoxaline-2-carbonyl)-amino]-propyl}-carbamicacid thiazol-5-ylmethyl ester.

The above mentioned compounds have been found to confer minimal or noresistance against HIV and are therefore useful alternatives forritonavir (RTV) as boosters of HIV inhibitors.

It has also been found that the above compounds are useful as boostingagents of other viral inhibitors such as for instance HCV and/or RSVinhibitors. The combination of said compounds and other drugs, such asHIV, HCV or RSV inhibitors is beneficial in that it permits theprovision of a therapy to infected patients which is safe, is effective,and allows a lower therapeutically effective dose of antivirals,compared to when such antivirals would be administered alone. A lowerdose is always desirable in terms of toxicity and pill burden, therebydiminishing the incidence of adverse effects and increasing treatmentcompliance, respectively. The combination of the compounds of formula(I) and (II) and HIV inhibitors, or other viral inhibitors, provides asynergistic effect on these antivirals upon administration of saidcombination to a patient in need thereof.

As used in the foregoing and hereinafter, the following definitionsapply unless otherwise noted.

Whenever the term “substituted” is used in defining the compounds of theinvention, it is meant to indicate that one or more hydrogens on theatoms mentioned or comprised in the expression using “substituted” isreplaced with a selection from the indicated group, provided that thesaid atoms' normal valency is not exceeded, and that the substitutionresults in a chemically stable compound, i.e. a compound that maintainsits structural and molecular identity in a useful degree of puritythrough a convenient amount of time. The convenient amount of time willdepend on the field of application.

The term halo(gen) is generic to fluoro, chloro, bromo and iodo.

As used herein “C₁₋₄ alkyl” as a group or part of a group definesstraight or branched chain saturated hydrocarbon radicals having from 1to 4 carbon atoms such as for example methyl, ethyl, 1-propyl, 2-propyl,1-butyl, 2-butyl, 2-methyl-1-propyl;

“C₁₋₆alkyl” encompasses C₁₋₄alkyl radicals and the higher homologuesthereof having 5 or 6 carbon atoms such as, for example, 1-pentyl,2-pentyl, 3-pentyl, 1-hexyl, 2-hexyl, 2-methyl-1-butyl,2-methyl-1-pentyl, 2-ethyl-1-butyl, 3-methyl-2-pentyl, and the like. Ofinterest amongst C₁₋₆alkyl is C₁₋₄alkyl especially isobutyl.

C₃₋₇cycloalkyl is generic to cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl and cycloheptyl.

“Heteroaryl” is art-recognized and refers to a monocyclic or bicyclicring system containing one or two five (5)- or six (6)-member (fused)aromatic rings; said ring system contains at least one hetero atomselected from nitrogen, oxygen or sulfur and said hetero atoms areoptionally substituted with C₁₋₆ alkyl.

It should be noted that the radical positions on any molecular moietyused in the definitions may be anywhere on such moiety as long as it ischemically stable.

Radicals used in the definitions of the variables include all possibleisomers unless otherwise indicated. For instance pyridyl includes2-pyridyl, 3-pyridyl and 4-pyridyl.

Whenever used hereinafter, the term “compounds of formula (I) forinstance”, “the present compounds”, “the compounds of the invention” orsimilar terms, it is meant to include the compounds of formula (I) andany subgroup thereof, the compounds as depicted in the Tables andExamples below, and the prodrugs, stereochemically isomeric forms,racemic mixtures, esters, addition salts, solvates, quaternary amines,N-oxides, metal complexes, and metabolites of any of the compoundsabove. One embodiment comprises the compounds of formula (I), or anysubgroup thereof specified herein, as well as the N-oxides, salts, aswell as possible stereoisomeric forms thereof.

Whenever used hereinafter, the term “HIV antiviral(s)” and “HIVinhibitor(s)” are interchangeable and have in the context of the currentdescription the same meaning.

The compounds of formula (I) may encompass centers of chirality in theirsubstituents and therefore exist as stereochemically isomeric forms. Theterms “stereochemically isomeric forms”, “stereoisomeric forms”, andequivalent terminology as used herein defines all the possible compoundsmade up of the same atoms bonded by the same sequence of bonds buthaving different three-dimensional structures which are notinterchangeable, which the compounds of formula (I) may possess.

With reference to the instances where (R) or (S), or alternativelyindicated by an asterisk (*), is used to designate the absoluteconfiguration of a chiral atom within a substituent, the designation isdone taking into consideration the whole compound and not thesubstituent in isolation.

Unless otherwise mentioned or indicated, the chemical designation of acompound encompasses the mixture of all possible stereochemicallyisomeric forms, which said compound may possess. Said mixture maycontain all diastereomers and/or enantiomers of the basic molecularstructure of said compound. All stereochemically isomeric forms of thecompounds of the present invention both in pure form or mixed with eachother are intended to be embraced within the scope of the presentinvention.

Pure stereoisomeric forms of the compounds and intermediates asmentioned herein are defined as isomers substantially free of otherenantiomeric or diastereomeric forms of the same basic molecularstructure of said compounds or intermediates. In particular, the term“stereoisomerically pure” concerns compounds or intermediates having astereoisomeric excess of at least 80% (i.e. minimum 90% of one isomerand maximum 10% of the other possible isomers) up to a stereoisomericexcess of 100% (i.e. 100% of one isomer and none of the other), more inparticular, compounds or intermediates having a stereoisomeric excess of90% up to 100%, even more in particular having a stereoisomeric excessof 94% up to 100% and most in particular having a stereoisomeric excessof 97% up to 100%. The terms “enantiomerically pure” and“diastereomerically pure” should be understood in a similar way, butthen having regard to the enantiomeric excess, and the diastereomericexcess, respectively, of the mixture in question.

Pure stereoisomeric forms of the compounds and intermediates of thisinvention may be obtained by the application of art-known procedures.For instance, enantiomers may be separated from each other by theselective crystallization of their diastereomeric salts with opticallyactive acids or bases. Examples thereof are tartaric acid,dibenzoyltartaric acid, ditoluoyltartaric acid and camphosulfonic acid.Alternatively, enantiomers may be separated by chromatographictechniques using chiral stationary phases. Said pure stereochemicallyisomeric forms may also be derived from the corresponding purestereochemically isomeric forms of the appropriate starting materials,provided that the reaction occurs stereospecifically. Preferably, if aspecific stereoisomer is desired, said compound will be synthesized bystereospecific methods of preparation. These methods will advantageouslyemploy enantiomerically pure starting materials.

The diastereomeric racemates of the compounds of the invention can beobtained separately by conventional methods. Appropriate physicalseparation methods that may advantageously be employed are, for example,selective crystallization and chromatography, e.g. columnchromatography.

For certain compounds of the invention, their prodrugs, N-oxides, salts,solvates, quaternary amines, or metal complexes, and the intermediatesused in the preparation thereof, the absolute stereochemicalconfiguration is not experimentally determined. A person skilled in theart is able to determine the absolute configuration of such compoundsusing art-known methods such as, for example, X-ray diffraction.

The present invention is also intended to include all isotopes of atomsoccurring on the present compounds. Isotopes include those atoms havingthe same atomic number but different mass numbers. By way of generalexample and without limitation, isotopes of hydrogen include tritium anddeuterium. Isotopes of carbon include C-13 and C-14.

The term “prodrug” as used throughout this text means thepharmacologically acceptable derivatives such as esters, amides andphosphates, such that the resulting in vivo biotransformation product ofthe derivative is the active drug as defined in the compounds of formula(I). The reference by Goodman and Gilman (The Pharmacological Basis ofTherapeutics, 8^(th) ed, McGraw-Hill, Int. Ed. 1992, “Biotransformationof Drugs”, p 13-15) describing prodrugs generally is herebyincorporated. Prodrugs preferably have excellent aqueous solubility,increased bioavailability and are readily metabolized into the activeinhibitors in vivo. Prodrugs of a compound of the present invention maybe prepared by modifying functional groups present in the compound insuch a way that the modifications are cleaved, either by routinemanipulation or in vivo, to the parent compound.

Preferred are pharmaceutically acceptable ester prodrugs that arehydrolysable in vivo and are derived from those compounds of formula (I)having a hydroxy or a carboxyl group. An in vivo hydrolysable ester isan ester, which is hydrolysed in the human or animal body to produce theparent acid or alcohol. Suitable pharmaceutically acceptable esters forcarboxy include C₁₋₆alkoxy-methyl esters for example methoxymethyl,C₁₋₆alkanoyloxymethyl esters for example pivaloyloxymethyl, phthalidylesters, C₃₋₈cycloalkoxycarbonyloxy-C₁₋₆alkyl esters for example1-cyclohexylcarbonyl-oxyethyl; 1,3-dioxolen-2-onylmethyl esters forexample 5-methyl-1,3-dioxolen-2-onylmethyl; andC₁₋₆alkoxycarbonyloxyethyl esters for example 1-methoxycarbonyloxyethylwhich may be formed at any carboxy group in the compounds of thisinvention.

An in vivo hydrolysable ester of a compound of the formula (I)containing a hydroxy group includes inorganic esters such as phosphateesters and α-acyloxyalkyl ethers and related compounds which as a resultof the in vivo hydrolysis of the ester breakdown to give the parenthydroxy group. Examples of α-acyloxyalkyl ethers include acetoxy-methoxyand 2,2-dimethylpropionyl-oxymethoxy. A selection of in vivohydrolysable ester forming groups for hydroxy include alkanoyl, benzoyl,phenylacetyl and substituted benzoyl and phenylacetyl, alkoxycarbonyl(to give alkyl carbonate esters), dialkylcarbamoyl andN-(dialkylaminoethyl)-N-alkylcarbamoyl (to give carbamates),dialkylamino-acetyl and carboxyacetyl. Examples of substituents onbenzoyl include morpholino and piperazino linked from a ring nitrogenatom via a methylene group to the 3- or 4-position of the benzoyl ring.

For therapeutic use, salts of the compounds of formula (I) are thosewherein the counter-ion is pharmaceutically acceptable. However, saltsof acids and bases which are non-pharmaceutically acceptable may alsofind use, for example, in the preparation or purification of apharmaceutically acceptable compound. All salts, whetherpharmaceutically acceptable or not are included within the ambit of thepresent invention.

The pharmaceutically acceptable acid and base addition salts asmentioned hereinabove are meant to comprise the therapeutically activenon-toxic acid and base addition salt forms which the compounds offormula (I) are able to form. The pharmaceutically acceptable acidaddition salts can conveniently be obtained by treating the base formwith such appropriate acid. Appropriate acids comprise, for example,inorganic acids such as hydrohalic acids, e.g. hydrochloric orhydrobromic acid, sulfuric, nitric, phosphoric and the like acids; ororganic acids such as, for example, acetic, propanoic, hydroxyacetic,lactic, pyruvic, oxalic (i.e. ethanedioic), malonic, succinic (i.e.butanedioic acid), maleic, fumaric, malic (i.e. hydroxybutanedioicacid), tartaric, citric, methane-sulfonic, ethanesulfonic,benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic,p-aminosalicylic, palmoic and the like acids.

Conversely said salt forms can be converted by treatment with anappropriate base into the free base form.

The compounds of formula (I) containing an acidic proton may also beconverted into their non-toxic metal or amine addition salt forms bytreatment with appropriate organic and inorganic bases. Appropriate basesalt forms comprise, for example, the ammonium salts, the alkali andearth alkaline metal salts, e.g. the lithium, sodium, potassium,magnesium, calcium salts and the like, salts with organic bases, e.g.the benzathine, N-methyl-D-glucamine, hydrabamine salts, and salts withamino acids such as, for example, arginine, lysine and the like.

The term “solvate” is used herein to describe a molecular complexcomprising i) the compounds of the invention as well as the saltsthereof, and ii) one or more pharmaceutically acceptable solventmolecules, for example, ethanol, isopropanol, 1-methoxy-2-propanol,methanol, acetone, dichloromethane, ethylacetate, anisol,tetrahydrofurane, or mesylate. The term “hydrate” is employed when saidsolvent is water.

The term “quaternary amine” as used hereinbefore defines the quaternaryammonium salts which the compounds of formula (I) are able to form byreaction between a basic nitrogen of a compound of formula (I) and anappropriate quaternizing agent, such as, for example, an optionallysubstituted alkylhalide, arylhalide or arylalkylhalide, e.g.methyliodide or benzyliodide. Other reactants with good leaving groupsmay also be used, such as alkyl trifluoromethanesulfonates, alkylmethanesulfonates, and alkyl p-toluene-sulfonates. A quaternary aminehas a positively charged nitrogen. Pharmaceutically acceptablecounterions include chloro, bromo, iodo, trifluoroacetate and acetate.The counterion of choice can be introduced using ion exchange resins.

The N-oxide forms of the present compounds are meant to comprise thecompounds of formula (I) wherein one or several nitrogen atoms areoxidized to the so-called N-oxide.

It will be appreciated that the compounds of formula (I) may have metalbinding, chelating, complex forming properties and therefore may existas metal complexes or metal chelates. Such metalated derivatives of thecompounds of formula (I) are intended to be included within the scope ofthe present invention.

Some of the compounds of formula (I) may also exist in their tautomericform. Such forms although not explicitly indicated in the above formulaare intended to be included within the scope of the present invention.

The compounds of formula (I) have two asymmetric centers as depicted bythe asterisk below:

Preferably the compounds of formula (I) have the stereochemistry asindicated in the structure of formula (I-a) below:

The compounds of formula (I) according to the present invention can beselected from any one of the following compounds of Table 1. Besides thesubstitution pattern (indicated by R, R₁, R₂) the LC-MS data (m/z (M+1)& retention time (R_(t)) is reported. The results and examples given arepresented to exemplify the invention and are not to be construed aslimiting the scope of the invention.

(I)

Comp. m/z No. R₁ R₂ R₃ (M + 1) Rt(min) C1 5-thiazolyl isobutyl

526 2.19 C2 5-thiazolyl isobutyl

522 2.29 C3 5-thiazolyl isobutyl

496 2.36 C4 5-thiazolyl isobutyl

550 2.49 C5 5-thiazolyl isobutyl

512 2.23 C6 5-thiazolyl isobutyl

500 2.26 C7 5-thiazolyl isobutyl

512 2.25 C8 5-thiazolyl isobutyl

483 3.84 C9 5-thiazolyl isobutyl

483 2.02 C10 5-thiazolyl isobutyl

496 2.36 C11 5-thiazolyl isobutyl

550 2.48 C12 5-thiazolyl isobutyl

534 2.32 C13 5-thiazolyl isobutyl

483 1.88 C14 5-thiazolyl isobutyl

471 2.13 C15 5-thiazolyl isobutyl

516 2.49 C16 5-thiazolyl isobutyl

516 2.41 C17 5-thiazolyl isobutyl

560 4.92 C18 5-thiazolyl isobutyl

560 2.45 C19 5-thiazolyl isobutyl

500 2.27 C20 3-pyridinyl isobutyl

477 1.79 C21 5-thiazolyl isobutyl

472 2.12 C22 5-thiazolyl 2,2- dimethylpropyl

497 4.05 C23 5-thiazolyl isobutyl

524 2.36 C24 5-thiazolyl isobutyl

517 3.88 C25 5-thiazolyl isobutyl

519 2.13 C26 5-thiazolyl 2-hydroxy-2- methyl-propyl

499 1.47 C27 5-thiazolyl cyclohexylmethyl

523 2.17

Preferred compound is compound C12.

In a preferred embodiment of the present invention there is provided acombination comprising (a) a compound of formula (I), a salt or astereoisomeric form thereof; and (b) an HIV antiviral, or apharmaceutically acceptable salt thereof; wherein the compound offormula (I) is C12.

In a preferred embodiment of the present invention there is provided acombination comprising (a) a compound of formula (I), a salt or astereoisomeric form thereof; and (b) an HIV antiviral, or apharmaceutically acceptable salt thereof; wherein the compound offormula (I) is C12 and wherein the HIV antiviral is selected from forinstance: nucleoside reverse transcriptase inhibitors (NRTIs), e.g.zidovudine (AZT), didanosine (ddI), zalcitabine (ddC), lamivudine (3TC),stavudine (d4T), emtricitabine (FTC), abacavir (ABC), apricitabine(AVX-754), elvucitabine (ACH-126,443), phosphazide, KP-1461, MIV-210,racivir (PSI-5004), and the like; or non-nucleoside reversetranscriptase inhibitors (NNRTIs) such as delavirdine (DLV), efavirenz(EFV), nevirapine (NVP), capravirine (CPV), calanolide A, dapivirine(TMC120), etravirine (TMC125), rilpivirine (TMC278), alovudine(MIV-310), UC-781, and the like; or nucleotide reverse transcriptaseinhibitors (NtRTIs), e.g. tenofovir and tenofovir disoproxil fumarate(TDF), and the like; or inhibitors of trans-activating proteins, such asTAT-inhibitors, e.g. RO-5-3335, BI-201; REV inhibitors; or proteaseinhibitors e.g. ritonavir (RTV), saquinavir (SQV), lopinavir (ABT-378 orLPV), indinavir (IDV), amprenavir (VX-478), TMC-126, nelfinavir(AG-1343), atazanavir (BMS-232632), darunavir (TMC-114) now marketed asPrezista™, SPI-256, fosamprenavir (GW433908 or VX-175), P-1946, MK-8122(PPL-100), tipranavir (PNU-140690), or a protease inhibitor with thechemical name(1-benzyl-3-{[2-(1-cyclopentyl-piperidin-4-ylamino)-benzothiazole-6-sulfonyl]-isobutyl-amino}-2-hydroxy-propyl)-carbamicacid hexahydro-furo[2,3-b]furan-3-yl ester, and the like; or viralintegrase inhibitors e.g. raltegravir (MK-518), elvitegravir (GS-9137;JTK-303), BMS-538,158, and the like; or entry inhibitors which comprisefusion inhibitors (e.g. T-20 or enfuvirtide, T-1249), attachmentinhibitors and co-receptor inhibitors; the latter comprise the CCR5antagonists and CXR4 antagonists (e.g. AMD-3100); examples of entryinhibitors are PRO-140, PRO-542, TBR-220 (TAK-220), TBR-652 (TAK-652),vicriviroc (SCH-417,690), TNX-355, maraviroc (UK-427,857), BMS-488,043,BMS-806; a maturation inhibitor for example is bevirimat (PA-457);ribonucleotide reductase inhibitors (cellular inhibitors), e.g.hydroxyurea and the like; or combinations of any of the above.

Most preferred embodiment is a combination comprising (a) a compoundC12, a salt or a stereoisomeric form thereof; and (b) an HIV antiviralselected from darunavir([(1R,5S,6R)-2,8-dioxabicyclo[3.3.0]oct-6-yl]N-[(2S,3R)-4-[(4-amino-phenyl)sulfonyl-(2-methylpropyl)amino]-3-hydroxy-1-phenyl-butan-2-yl]-carbamate)or a compound with the chemical name(1-benzyl-3-{[2-(1-cyclopentyl-piperidin-4-ylamino)-benzothiazole-6-sulfonyl]-isobutyl-amino}-2-hydroxy-propyl)-carbamicacid hexahydro-furo[2,3-b]furan-3-yl ester.

In one embodiment of the present invention there is provided a processfor preparing a combination as described herein, comprising the step ofcombining a compound of formula (I) or a pharmaceutically acceptablesalt thereof, and an HIV antiviral, or a pharmaceutically acceptablesalt thereof. An alternative embodiment of this invention provides aprocess wherein the combination comprises one or more additional agentas described herein.

The combinations of the present invention may be used as medicaments.Said use as a medicine or method of treatment comprises the systemicadministration to HIV-infected subjects of an amount effective to combatthe conditions associated with HIV. Consequently, the combinations ofthe present invention can be used in the manufacture of a medicamentuseful for treating, preventing or combating infection or diseaseassociated with HIV infection in a mammal.

In one embodiment of the present invention there is provided apharmaceutical composition comprising a combination according to any oneof the embodiments described herein and a pharmaceutically acceptableexcipient. In particular, the present invention provides apharmaceutical composition comprising (a) a therapeutically effectiveamount of a compound of formula (I) or a pharmaceutically acceptablesalt thereof, (b) a therapeutically effective amount of an HIVantiviral, or a pharmaceutically acceptable salt thereof, and (c) apharmaceutically acceptable excipient. Optionally, the pharmaceuticalcomposition further comprises an additional agent selected from an HIVantiviral.

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients, as well as any productwhich results, directly or indirectly, from the combination of thespecified ingredients.

The term “therapeutically effective amount” as used herein means thatamount of active compound or component or pharmaceutical agent thatelicits the biological or medicinal response in a tissue, system, animalor human that is being sought, in the light of the present invention, bya researcher, veterinarian, medical doctor or other clinician, whichincludes alleviation of the symptoms of the disease being treated. Sincethe instant invention refers to combinations comprising two or moreagents, the “therapeutically effective amount” is that amount of theagents taken together so that the combined effect elicits the desiredbiological or medicinal response. For example, the therapeuticallyeffective amount of a composition comprising (a) the compound of formula(I) and (b) an HIV antiviral, would be the amount of the compound offormula (I) and the amount of the HIV antiviral that when taken togetherhave a combined effect that is therapeutically effective.

The pharmaceutical composition can be prepared in a manner known per seto one of skill in the art. For this purpose, at least one of a compoundof formula (I) or any subgroup thereof, and an HIV antiviral, togetherwith one or more solid or liquid pharmaceutical excipients and, ifdesired, in combination with other pharmaceutical active compounds, arebrought into a suitable administration form or dosage form which canthen be used as a pharmaceutical in human medicine or veterinarymedicine.

In one embodiment the combinations of the present invention may also beformulated as a combined preparation for simultaneous, separate orsequential use in HIV therapy, as applicable. In such a case, thecompound of general formula (I) or any subgroup thereof, is formulatedin a pharmaceutical composition containing other pharmaceuticallyacceptable excipients, and the appropriate HIV antiviral is formulatedseparately in a pharmaceutical composition containing otherpharmaceutically acceptable excipients. Conveniently, these two separatepharmaceutical compositions can be part of a kit for simultaneous,separate or sequential use.

Thus, the individual components of the combination of the presentinvention can be administered separately at different times during thecourse of therapy or concurrently in divided or single combinationforms. The present invention is therefore to be understood as embracingall such regimes of simultaneous or alternating treatment and the term“administering” is to be interpreted accordingly. In a preferredembodiment, the separate dosage forms are administered aboutsimultaneously.

The compositions or products comprising a combination of the presentinvention, whether co-formulated in a single formulation or formulatedfor simultaneous, separate or sequential use, may be administered orally(including suspensions, capsules, tablets, sachets, solutions,suspensions, emulsions), sublingually, parenterally (includingsubcutaneous injections, intravenous, intramuscular, intradermalinjection or infusion techniques), topically, rectally (includingsuppositories), vaginally, via an implanted reservoir, in dosage unitformulations containing conventional non-toxic pharmaceuticallyacceptable carriers, adjuvants and vehicles.

For an oral administration form, the compositions of the presentinvention can be mixed with suitable additives, such as excipients,stabilizers or inert diluents, and brought by means of the customarymethods into the suitable administration forms, such as tablets, coatedtablets, hard capsules, aqueous, alcoholic, or oily solutions. Examplesof suitable inert carriers are gum arabic, magnesia, magnesiumcarbonate, potassium phosphate, lactose, glucose, or starch, inparticular, corn starch. In this case, the preparation can be carriedout both as dry and as moist granules. Suitable oily excipients orsolvents are vegetable or animal oils, such as sunflower oil or codliver oil. Suitable solvents for aqueous or alcoholic solutions arewater, ethanol, sugar solutions, or mixtures thereof. Polyethyleneglycols and polypropylene glycols are also useful as further auxiliariesfor other administration forms. As immediate release tablets, thesecompositions may contain microcrystalline cellulose, dicalciumphosphate, starch, magnesium stearate and lactose and/or otherexcipients, binders, extenders, disintegrants, diluents and lubricantsknown in the art.

The oral administration of a combination of the present invention issuitably accomplished by uniformly and intimately blending together asuitable amount of each component in the form of a powder, optionallyalso including a finely divided solid carrier, and encapsulating theblend in, for example, a hard gelatin capsule. The solid carrier caninclude one or more substances which act as binders, lubricants,disintegrating agents, coloring agents, and the like. Suitable solidcarriers include, for example, calcium phosphate, magnesium stearate,talc, sugars, lactose, dextrin, starch, gelatin, cellulose,polyvinyl-pyrrolidine, low melting waxes and ion exchange resins.

Oral administration of a combination of the present invention can alsobe accomplished by preparing capsules or tablets containing the desiredamount of the compound of formula (I) only, optionally blended with asolid carrier as described above, and capsules containing the desiredamount of the HIV antiviral only. Compressed tablets containing thecompound of formula (I) can be prepared by uniformly and intimatelymixing the active ingredient with a solid carrier such as describedabove to provide a mixture having the necessary compression properties,and then compacting the mixture in a suitable machine to the shape andsize desired. Molded tablets maybe made by molding in a suitablemachine, a mixture of powdered the compound of formula (I) moistenedwith an inert liquid diluents. Oral administration can also beaccomplished by preparing compressed or molded tablets containing thecompound of formula (I) as just described, the tablets of suitable sizefor insertion into standard capsules (e.g., hard gelatin capsules), andthen inserting the tablets into capsules containing a suitable amount ofHIV antiviral powder.

For subcutaneous or intravenous administration, the active components ofthe compositions, if desired with the substances customary thereforesuch as solubilizers, emulsifiers or further auxiliaries, are broughtinto solution, suspension, or emulsion. The components of thecompositions can also be lyophilized and the lyophilizates obtainedused, for example, for the production of injection or infusionpreparations. Suitable solvents are, for example, water, physiologicalsaline solution or alcohols, e.g. ethanol, propanol, glycerol, inaddition also sugar solutions such as glucose or mannitol solutions, oralternatively mixtures of the various solvents mentioned. The injectablesolutions or suspensions may be formulated according to known art, usingsuitable non-toxic, parenterally-acceptable diluents or solvents, suchas mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodiumchloride solution, or suitable dispersing or wetting and suspendingagents, such as sterile, bland, fixed oils, including synthetic mono- ordiglycerides, and fatty acids, including oleic acid.

The pharmaceutical compositions of this invention may also beadministered topically, especially when the target of treatment includesareas or organs readily accessible by topical application, includingdiseases of the eye, the skin, or the lower intestinal tract. Suitabletopical formulations are readily prepared for each of these areas ororgans. Topical application for the lower intestinal tract may beeffected in a rectal suppository formulation (see below) or in asuitable enema formulation. Topically-transdermal patches may also beused.

For topical applications, the pharmaceutical compositions may beformulated in a suitable ointment containing the active componentssuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds of this invention include, but are notlimited to, mineral oil, liquid petrolatum, white petrolatum, propyleneglycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax andwater. Alternatively, the pharmaceutical compositions may be formulatedin a suitable lotion or cream containing the active components suspendedor dissolved in one or more pharmaceutically acceptable carriers.Suitable carriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water.

When rectally administered in the form of suppositories, theseformulations may be prepared by mixing the individual components of acomposition according to the invention with a suitable non-irritatingexcipient, such as cocoa butter, synthetic glyceride esters orpolyethylene glycols, which are solid at ordinary temperatures, butliquidify and/or dissolve in the rectal cavity to release the drug.

In another embodiment of the method of the invention, the administrationmay be performed with food (e.g., a high-fat meal) or without food. Theterm “with food” means the consumption of a meal either during or nomore than about one hour before or after administration of a one or bothcomponents of the combination according to the invention.

In one embodiment, the combinations of the present invention contain anamount of a compound of formula (I), or a pharmaceutically acceptablesalt thereof, which is sufficient to clinically improve thebioavailability of the HIV inhibitor or antiviral relative to thebioavailability when said HIV inhibitor or antiviral is administeredalone.

In another embodiment, the combinations of the present invention containan amount of a compound of formula (I), or a pharmaceutically acceptablesalt thereof, which is sufficient to increase at least one of thepharmacokinetic variables of the HIV inhibitors selected from t_(1/2),C_(min), C_(max), C_(ss), AUC at for instance 12 hours, or AUC at forinstance 24 hours, relative to said at least one pharmacokineticvariable when said HIV inhibitor is administered alone.

A further embodiment relates to a method for improving thebioavailability of an HIV inhibitor comprising administering to anindividual in need of such improvement a combination as defined herein,comprising a therapeutically effective amount of each component of saidcombination.

In a further embodiment, the invention relates to the use of a compoundof formula (I) or a pharmaceutically acceptable salt thereof, as animprover of at least one of the pharmacokinetic variables of an HIVinhibitor selected from t_(1/2), C_(min), C_(max), C_(ss), AUC at forinstance 12 hours, or AUC at for instance 24 hours; with the provisothat said use is not practiced in the human or animal body.

The term “individual” as used herein refers to an animal, preferably amammal, most preferably a human, who has been the object of treatment,observation or experiment.

Bioavailability is defined as the fraction of administered dose reachingsystemic circulation. t_(1/2) represents the half life or time taken forthe plasma concentration to fall to half its original value. C_(ss) isthe steady state concentration, i.e. the concentration at which the rateof input of drug equals the rate of elimination. C_(min) is defined asthe lowest (minimum) concentration measured during the dosing interval.C_(max), represents the highest (maximum) concentration measured duringthe dosing interval. AUC is defined as the area under the plasmaconcentration-time curve for a defined period of time for instance at 12hrs or 24 hrs.

The combinations of this invention can be administered to humans indosage ranges specific for each component comprised in saidcombinations. The components comprised in said combinations can beadministered together or separately. The HIV inhibitors, and thecompound of formula (I) or a pharmaceutically acceptable salt or esterthereof, may have dosage levels of the order of 0.02 to 5.0grams-per-day.

When the HIV inhibitor or antiviral and the compound of formula (I) areadministered in combination, the weight ratio of the HIV inhibitor tothe compound of formula (I) is suitably in the range of from about 40:1to about 1:15, or from about 30:1 to about 1:15, or from about 15:1 toabout 1:15, typically from about 10:1 to about 1:10, and more typicallyfrom about 8:1 to about 1:8. Also useful are weight ratios of the HIVinhibitor to compound of formula (I) ranging from about 6:1 to about1:6, or from about 4:1 to about 1:4, or from about 3:1 to about 1:3, orfrom about 2:1 to about 1:2, or from about 1.5:1 to about 1:1.5. In oneaspect, the amount by weight of the HIV inhibitor is equal to or greaterthan that of the compound of formula (I), wherein the weight ratio ofthe HIV inhibitor to the compound of formula (I) is suitably in therange of from about 1:1 to about 15:1, typically from about 1:1 to about10:1, and more typically from about 1:1 to about 8:1. Also useful areweight ratios of the HIV inhibitor to the compound of formula (I)ranging from about 1:1 to about 6:1, or from about 1:1 to about 5:1, orfrom about 1:1 to about 4:1, or from about 3:2 to about 3:1, or fromabout 1:1 to about 2:1 or from about 1:1 to about 1.5:1.

According to one embodiment, the HIV inhibitor and the compound offormula (I) may be co-administered once or twice a day, once, twice,three, four, fives or six times a week, preferably orally, wherein theamount of the HIV inhibitor per dose is from about 10 to about 2500 mg,and the amount of the compound of formula (I) per dose is from 10 toabout 2500 mg. In another embodiment, the amounts per dose for once ortwice daily co-administration are from about 50 to about 1500 mg of theHIV inhibitor and from about 50 to about 1500 mg of the compound offormula (I). In still another embodiment, the amounts per dose for thedaily or weekly co-administration are from about 100 to about 1000 mg ofthe HIV inhibitor and from about 100 to about 800 mg of the compound offormula (I). In yet another embodiment, the amounts per dose for thedaily or weekly co-administration are from about 150 to about 800 mg ofthe HIV inhibitor and from about 100 to about 600 mg of the compound offormula (I). In yet another embodiment, the amounts per dose for thedaily or weekly co-administration are from about 200 to about 600 mg ofthe HIV inhibitor and from about 100 to about 400 mg of the compound offormula (I). In yet another embodiment, the amounts per dose for thedaily or weekly co-administration are from about 200 to about 600 mg ofthe HIV inhibitor and from about 20 to about 300 mg of the compound offormula (I). In yet another embodiment, the amounts per dose for thedaily or weekly co-administration are from about 100 to about 400 mg ofthe HIV inhibitor and from about 40 to about 100 mg of the compound offormula (I).

Exemplary combinations of the HIV inhibitor (mg)/compound of formula (I)(mg) for twice daily dosage include 50/100, 100/100, 150/100, 200/100,250/100, 300/100, 350/100, 400/100, 450/100, 50/133, 100/133, 150/133,200/133, 250/133, 300/133, 50/150, 100/150, 150/150, 200/150, 250/150,50/200, 100/200, 150/200, 200/200, 250/200, 300/200, 50/300, 80/300,150/300, 200/300, 250/300, 300/300, 200/600, 400/600, 600/600, 800/600,1000/600, 200/666, 400/666, 600/666, 800/666, 1000/666, 1200/666,200/800, 400/800, 600/800, 800/800, 1000/800, 1200/800, 200/1200,400/1200, 600/1200, 800/1200, 1000/1200, and 1200/1200. Other exemplarycombinations of the HIV inhibitor (mg)/compound of formula (I) (mg) fortwice daily dosage include 1200/400, 800/400, 600/400, 400/200, 600/200,600/100, 500/100, 400/50, 300/50, and 200/50.

It will be understood, however, that specific dose level and frequencyof dosage for any particular patient may be varied and will depend upona variety of factors including the activity of the specific compoundemployed, the metabolic stability and length of action of that compound;the age, body weight, general health, sex and diet of the patient; modeand time of administration, rate of excretion, drug combination, theseverity of the particular condition, and the type of patient undergoingtherapy.

In one embodiment of the present invention there is provided an articleof manufacture comprising a composition effective to treat an HIVinfection; and packaging material comprising a label which indicatesthat the composition can be used to treat infection by HIV; wherein thecomposition comprises the combination as described herein.

EXAMPLES

The results and examples given are presented to exemplify the inventionand are not to be construed as limiting the scope of the invention.

The compounds according to the invention have been tested in various invitro assays for which the results are listed in the Tables hereafter.

Assay 1 &2: Antiviral Activity/Toxicity

The compounds of the present invention were tested for antiviralactivity in a cellular assay, which was performed according to thefollowing procedure.

The human T-cell line MT4 is engineered with Green Fluorescent Protein(GFP) and an HIV-specific promoter, HIV-1 long terminal repeat (LTR).This cell line is designated MT4 LTR-EGFP, and can be used for the invitro evaluation of anti-HIV activity of investigational compounds. InHIV-1 infected cells, the Tat protein is produced which upregulates theLTR promotor and finally leads to stimulation of the GFP reporterproduction, allowing measuring ongoing HIV-infection fluorometrically.

Analogously, MT4 cells are engineered with GFP and the constitutionalcytomegalovirus (CMV) promotor. This cell line is designated MT4CMV-EGFP, and can be used for the in vitro evaluation of cytotoxicity ofinvestigational compounds. In this cell line, GFP levels are comparablyto those of infected MT4 LTR-EGFP cells. Cytotoxic investigationalcompounds reduce GFP levels of mock-infected MT4 CMV-EGFP cells.

Effective concentration values such as 50% effective concentration(EC₅₀) can be determined and are usually expressed in μM. An EC₅₀ valueis defined as the concentration of test compound that reduces thefluorescence of HIV-infected cells by 50%. The 50% cytotoxicconcentration (CC₅₀ in μM) is defined as the concentration of testcompound that reduces fluorescence of the mock-infected cells by 50%.The ratio of CC₅₀ to EC₅₀ is defined as the selectivity index (SI) andis an indication of the selectivity of the anti-HIV activity of theinhibitor. The ultimate monitoring of HIV-1 infection and cytotoxicityis done using a scanning microscope. Image analysis allows verysensitive detection of viral infection. Measurements are done beforecell necrosis, which usually takes place about five days afterinfection, in particular measurements are performed three days afterinfection.

Table 2 lists pEC₅₀ values against the wild-type HIV-1 IIIB strain aswell as pCC50 values for a selected number of compounds of theinvention. A pEC₅₀ value corresponds to −log₁₀(EC₅₀). A pCC₅₀ valuecorresponds to −log₁₀(CC₅₀).

Listed are compounds having a pEC₅₀ value of less than 4.00 to maximum4.50 pEC₅₀. Darunavir, a commercially available HIV protease inhibitor,has an pEC₅₀ of 8.17. The pEC₅₀ range of <4 to 4.50, when compared to8.17, is significantly lower in terms of antiviral activity, thereforedemonstrating that the compounds of the present invention confer minimalor no resistance to HIV.

Equally, the toxicity values reported for the compounds of the presentinvention, in a range of less than 4.00 to maximum 4.46 pCC₅₀,demonstrate the low or minimal toxicity of these compounds.

TABLE 2 antiviral activity (pEC50) and cytotoxicity (pCC50) of selectedtest compounds. Compound Antiviral Activity IIIB Toxicity MT4 Nr. pEC₅₀pCC₅₀ C3 4.28 4.15 C4 4.50 4.40 C5 4.07 4.06 C6 4.10 <4.00 C7 4.08 4.15C8 <4.00 <4.00 C9 <4.00 <4.00 C10 <4.00 <4.00 C11 4.42 4.46 C12 4.224.21 C13 <4.00 <4.00 C14 4.10 4.05 C15 4.30 4.37 C16 4.41 4.39 C17 4.384.44 C18 4.41 4.38 C19 <4.00 <4.00 C20 <4.00 <4.00 C1 4.32 4.24 C2 4.354.34 C21 <4.00 <4.00 C22 <4.00 <4.00 C23 4.29 4.26 C24 4.01 <4.00 C254.16 4.11 C26 <4.00 <4.00 C27 4.13 4.08Assay 3: Metabolic Stability of Test Compounds (HLM15′)

Sub-cellular tissue preparations are made according to Gorrod et al.(Xenobiotica, 5, pp. 453-462 (1975)) by centrifugal separation aftermechanical homogenization of tissue. Human liver tissue is rinsed inice-cold 0.1 M Tris-HCl (pH 7.4) buffer to wash excess blood. Tissue isthen blotted dry, weighed and chopped coarsely using surgical scissors.The tissue pieces are homogenized in 3 volumes of ice-cold 0.1 Mphosphate buffer (pH 7.4) using either a Potter-S (Braun, Italy)equipped with a Teflon pestle or a Sorvall Omni-Mix homogenizer, for7×10 sec. In both cases, the vessel is kept in/on ice during thehomogenization process. Tissue homogenates are centrifuged at 9000×g for20 min at 4° C. using a Sorvall centrifuge or Beckman Ultracentrifuge.The resulting supernatant can be stored at −80° C. and is designated‘S9’. The S9 fraction is centrifuged at 100,000×g for 60 min at 4° C.using a Beckman ultracentrifuge. The resulting supernatant is carefullyaspirated, aliquoted and designated ‘cytosol’. The pellet isre-suspended in 0.1 M phosphate buffer (pH 7.4) in a final volume of 1ml per 0.5 g original tissue weight and designated ‘microsomes’. Allsub-cellular fractions are aliquoted, immediately frozen in liquidnitrogen and stored at −80° C. until use.

Test compounds and a NADPH-generating system were added to human livermicrosomes (‘microsomes’ fraction, protein concentration 1 mg/ml)suspended in 0.1 M phosphate buffer (pH=7.4), to get final reactionmixture concentrations of 5 μM test compound, 0.8 mMD-Glucose-6-phosphate, 0.8 mM MgCl₂ and 0.8 U/ml of Glucose-6-phosphatedehydrogenase. Heat-inactivated (10 min at 95° C.) ‘S9’ or microsomeswere used for blank experiments. Reaction mixtures were incubated at 37°C. for 5 min, after which the reaction was started by the addition of0.8 mM β-NADP. The reaction was incubated for 0 or 15 min. Next, thereaction was stopped by addition of 2 volumes of DMSO (or acetonitrile).Samples were centrifuged (10 min, 900×g) and analyzed by LC-MS. Table 3summarizes the results.

TABLE 3 Metabolisation of test compound by human liver microsomes after15 min. Compound % recovery Nr. HLM15 C3 54 C4 57 C5 83 C6 81 C7 56 C881 C9 55 C10 47 C11 52 C12 67 C13 69 C14 24 C15 57 C16 59 C17 68 C18 57C19 69 C20 37 C1 56 C2 67 C21 62 C22 77 C23 72 C24 56 C25 63 C26 92 C2798Assay 4: CYP450 Inhibition

Inhibition of the metabolism of test compounds by different CYP P450isoenzymes was determined using E. coli expressed proteins (3A4, 2C9,2D6, 1A2, and 2C19) that convert their specific substrates into afluorescent molecule (Table 6). This fluorescent molecule was measuredusing a fluorescent plate reader (Victor2 (Wallac) or Fluoroskan(Labsystems)). Compounds inhibiting the enzymatic reaction will resultin a decrease of fluorescent signal. CYP P450 enzymes were prepared inhouse or commercially bought and stored at −80° C. Table 4 summarizesthe results.

TABLE 4 Inhibition of CYP450 isoenzymes by test compounds. CYP P450isozymes 3A4- BFC 3A4- 3A4- 2C9- 2D6- 1A2- 2C19- Comp. % BQ DBF MFC AMMCCEC CEC Nr. inh. % inh. % inh. % inh. % inh. % inh. % inh. C3 95 85 9799 49 20 90 C4 95 85 99 99 79 62 92 C5 92 82 92 94 41 14 86 C6 82 83 9296 65 41 94 C7 92 80 94 97 45 13 89 C8 91 79 91 95 39 4 76 C9 91 78 8985 66 4 64 C10 95 79 93 96 62 33 92 C11 94 78 99 97 86 45 100 C12 70 7993 92 0 21 81 C13 81 84 94 95 64 8 67 C14 84 80 91 98 67 56 87 C15 75 8092 97 41 51 92 C16 71 82 92 97 43 50 90 C17 57 81 93 98 46 55 87 C18 8384 94 98 64 63 93 C19 58 72 92 93 53 43 77 C20 88 66 85 63 13 6 41 C1 7780 95 97 73 36 94 C2 100 79 96 96 102 40 98 C21 87 81 95 95 62 43 75 C2293 74 89 69 62 0 60 C23 94 73 92 89 65 22 77 C24 101 82 89 92 96 13 86C25 100 79 89 95 91 25 97 C26 95 77 84 82 30 0 35 C27 101 81 90 84 86 1096

TABLE 5 Conversions mediated by the respective E. coli expressed CYPP450 isoenzymes Substrate Enzyme Fluorescent molecule BFC:7-Benzyloxytrifluoro- CYP3A4 7-HFC: 7-Hydroxy- methyl coumarintrifluoromethyl coumarin BQ: 7-benzyloxyquinoline CYP3A4 7-HQ:7-hydroxyquinoline DBF: dibenzylfluorescein CYP3A4 fluorescein MFC7-Methoxy-4-trifluoro- CYP2C9 7-HFC: 7-Hydroxy- methyl coumarintrifluoromethyl coumarin AMMC: 3-[2-(N,N-diethyl-N- CYP2D6 AHMC:3-[2-(N,N- methylamino)ethyl]-7- diethylamino)ethyl]-7-methoxy-4-methylcoumarin hydroxy-4-methylcoumarin hydrochloride CEC:7-ethoxy-3-cyanocoumarin CYP1A2 CHC: 3-cyano-7- hydroxycoumarin CEC:7-ethoxy-3-cyanocoumarin CYP2C19 CHC: 3-cyano-7- hydroxycoumarin

The assay was performed in black 96 well Costar plates. Test compoundswere added to a CYP P450 enzyme solution in the presence of an NADPHgenerating system. After 5 min of preincubation at 37° C., the freshlyprepared, phosphate buffered (pH 7.4) substrate solution was added.Known CYP P450 inhibitors were used as positive controls, negativecontrols were run without CYP P450 enzyme. For final reaction mixtureconcentrations, see Table 6. Reaction mixtures were incubated at 37° C.for 30 min (CYP3A4-BFC), 30 min (CYP3A4-BQ), 10 min (CYP3A4-DBF), 15 min(CYP1A2-CEC), 30 min (CYP2C9-MFC, CYP2C19-CEC), or 45 min (CYP2D6-AMMC),respectively. Then, the reaction was stopped by the addition of 200 μlacetonitrile, and the fluorescent signal was detected.

TABLE 6 Final reaction mixture concentrations for CYP P450 isoenzymeinhibition assay. CYP3A4- CYP3A4- CYP3A4- CYP2C9- CYP2D6- CYP1A2-CYP2C19- BFC BQ DBF MFC AMMC CEC CEC Test compound 10 μM 10 μM 10 μM 10μM 10 μM 10 μM 10 μM NADPH Glucose-6- 3.3 mM 3.3 mM 3.3 mM 3.3 mM 0.41mM 3.3 mM 3.3 mM generating Phosphate system Glucose-6- 0.4 U/ml 0.4U/ml 0.4 U/ml 0.4 U/ml 0.4 U/ml 0.4 U/ml 0.4 U/ml Phosphate dehydro-genase MgCl₂ 3.3 mM 3.3 mM 3.3 mM 3.3 mM 0.41 mM 3.3 mM 3.3 mM NADP 1.3mM 1.3 mM 1.3 mM 1.3 mM 8.2 μM 1.3 mM 1.3 mM CYP P450 enzyme 83 nM 20 nM5 nM 60 nM 42 nM 5 nM 2.5 nM Substrate 150 μM 60 μM 1 μM 200 μM 3 μM 5μM 25 μM

Calculation of CYP P450 isoenzyme inhibition (% inh.):% activity=(100/(average positive control−average negativecontrol))×(average sample−average negative control)% inhibition=100−% activityAssay 5: % Metabolic Blocking: Inhibition of TMC114 Metabolisation

Darunavir (TMC 114 currently marketed as Prezista™) and tested boostercompounds were added to human liver microsomes (‘microsomes’ fraction,protein concentration 1 mg/ml) suspended in potassium phosphate buffer(pH=7.4), to get final reaction mixture concentrations of 3 μM darunavirand 3 μM test compound. In the non-boosted parallel reactions, testcompound was not added.

Boiled human liver microsomes were used for blank experiments. Afteraddition (in a 1:3 (v/v) ratio) of a NADPH generating mixture consistingof β-nicotinamide adenine dinucleotide phosphate (β-NADP, 0.5 mg/ml,653.2 μM), D-Glucose-6-phosphate (2 mg/ml, 7.1 mM), Glucose-6-phosphatedehydrogenase (1.5 U/ml) in 2% NaHCO₃, the reaction mixture wasincubated at 37° C. for 30 or 120 minutes after which the reaction wasstopped by increasing the temperature to 95° C. Darunavir concentrationswere determined using HPLC-MS. The control value, obtained when thepercentage TMC114 remaining is determined in the non boosted reaction(=absence of test compound), is 12% (median value of 10 experiments).Table 7 summarizes the results.

TABLE 7 Stability of darunavir in the presence of human liver microsomesand 3 μM tested booster compound % Stability of darunavir (% Compoundrecovery of darunavir after 120′) Nr. @ 3 μM booster C3 82 C4 83 C5 86C6 87 C7 95 C8 80 C9 25 C10 NA C11 108 C12 93 C13 72 C14 73 C15 119 C1686 C17 97 C18 82 C19 103 C20 41 C1 85 C2 100 C21 73 C22 58 C23 89 C24 87C25 104 C26 39 C27 85Booster Influence on Key Pharmacokinetic Parameters of Darunavir

To test the “boosting capacity” (=ability of the compounds to enhancethe pharmacokinetics of darunavir) in vivo, representative examplecompound C12 was given orally (in a suitable vehicle like for examplePEG400- or PEG400/30% saline or HpβCD) to a group (n=3) of fed, maleBeagle dogs, at a dose of 5 mg/kg body weight, 15 minutes prior toadministration of 5 mg/kg body weight darunavir. Oral dosing was done bygavage. At all times, the dogs were given free and continuous access towater. Blood samples were collected from the jugular vein at 0(=predose), 0.5, 1, 2, 4, 7, and 24 h after dose administration. Thesamples were centrifuged at 1900×g for 10 min at 5° C. to allow plasmaseparation. Separated plasma was stored in the freezer within two hoursafter blood sampling. At all times, blood and plasma samples were placedon melting ice and protected from light. Individual plasma samples wereanalyzed for darunavir and booster compound by means of LC-MS/MS.Pharmacokinetic parameters for darunavir were calculated usingnoncompartemental Analysis, WinNonLin software Version 5.0, Pharsight,and are listed in Table 8. Values listed are the average of 3 dogs. Foldchange (FC) values indicate the difference with the control experimentin which only 5 mg/kg darunavir was given.

TABLE 8 Booster influence on key pharmacokinetic parameters ofdarunavir. Compound AUC C_(max) C_(7h) FC FC FC Nr. ng · h/mL ng/mLng/mL AUC C_(max) C_(7 h) C12 1574 892 24 3 2 8

Experimental

Reagents were purchased from commercial sources and were used asreceived. Thin layer chromatography was performed on silica gel 60 F₂₅₄plates (Merck). LC-MS analysis was done using either one of thefollowing methods. Data for compounds C1-27 are listed in Table 1 (seeabove)

LCMS-Method 1

HPLC-system: Waters Alliance 2695 (pump+auto sampler), Waters 996 (Photodiode array-Detector)

Column: Waters XTerra MS C18 2.5 μm 50×4.6 mm

Temperature: 55° C.

Mobile phase: A: 10 mM HCOONH₄+0.1% HCOOH in H₂O

-   -   B: CH₃CN

Gradient: Omin: 15% B, 3 min: 95% B, 4.2 min: 95% B

Equilibration time: 1.2 min

Flow: 2 ml/min

Injection volume: 5 μl of a 0.5 mg/ml solution

MS-detector: Waters ZQ

Ionisation: electrospray in positive and negative mode

LCMS-Method 2

HPLC-system: Waters Alliance 2790 (pump+autosampler), Waters 996 (Photodiode array-detector)

Column: Waters SunFire C18 3.5 μm 100×4.6 mm

Temperature: 55° C.

Mobile phase: A: 10 mM NH₄OOCH+0.1% HCOOH in H2O

-   -   B: acetonitril

Gradiënt: 0 min: 5% B, 5.4 min: 95% B, 7.2 min: 95% B

Equilibration time: 1.8 min

Flow: 1.5 ml/min

Injection volume: 5 μl of a 0.5 mg/ml solution

MS-detector: Waters LCT

Ionisation: electrospray in positive and negative mode

NMR spectra were recorded on a Bruker Avance 400 spectrometer, operatingat 400 MHz. Chemical shifts are given in ppm and J values in Hz.Multiplicity is indicated using the following abbreviations: d fordoublet, t for a triplet, m for a multiplet, etc. Compound names weregenerated using Chemdraw Ultra, version 9.0.7 (CambridgeSoft).

Chemistry

The compounds of formula (I) were prepared according the general methodprovided below exemplified by the detailed procedures described for thesynthesis of C12.

A solution of 1 (1.0 eq., 106 mmol, 28.0 g) and isobutylamine (1060mmol, 10 eq., 106 mL) in isopropanol (200 mL) was stirred at 80° C. for3 h. The solvent was evaporated under reduced pressure and the crudeproduct 2 was used as such in the next step.

A solution of 2 (32.53 g, 97 mmol, 1.0 eq.), quinoxaline-2-carbonylchloride (18.62 g, 97 mmol, 1.0 eq.) and triethylamine (40.4 mL, 291mmol, 3.0 eq.) in THF (200 mL) was stirred at room temperature for 2 h.Water was added and extraction was carried out with CH₂Cl₂. The combinedorganic phases were washed twice with saturated aqueous NaHCO₃ solution,dried with MgSO₄ and concentrated in vacuo. The residue was purified bycolumn chromatography on silica gel (0-3% MeOH in CH₂Cl₂) to give 3(46.69 g, yield=98%).

A solution of 3 (19.58 g, 40 mmol, 1.0 eq.), chloro-trimethyl-silane(12.95 g, 120 mmol, 3.0 eq.), sodium iodide (23.83 g, 160 mmol, 4.0 eq.)and TBAF (199 mL, 200 mmol, 5.0 eq., 1.0 M solution in THF) inacetonitrile (150 mL) was stirred at RT for 1 h. Water was added andextraction was carried out with CH₂Cl₂. The combined organic phases weredried with MgSO₄ and concentrated in vacuo. The crude product was usedas such in the next step.

A solution of 4 (36.0 g, 92 mmol, 1.0 eq.), 5 (32.4 g, 127 mmol, 1.38eq.) and triethylamine (18.28 mL, 131 mmol, 1.43 eq.) in acetonitrile(500 mL) was stirred at room temperature for 2 h. Water was added andextraction was carried out with CH₂Cl₂. The combined organic phases werewashed twice with saturated aqueous NaHCO₃ solution, dried with MgSO₄and concentrated in vacuo. The residue was purified by columnchromatography on silica gel (0-2% NH₃ (7M solution in MeOH) in CH₂Cl₂)to give 6. A second purification by column chromatography on silica gel(EtOAc) was necessary to remove residual TBAF and to give pure 6 (═C12)(38.2 g, yield=78%, purity (LC)=94%). The NMR spectrum showed presenceof two rotamers present in a 2-1 ratio. ¹H-NMR (CDCl₃), major rotamer:9.28 ppm (s, 1H), 8.78 (s, 1H), 8.21 ppm (dd, 1H, J=8.41, J=2.2), 8.09(m, 1H), 7.92-7.8 (m, 2H); 7.77 (s, 1H); 7.3-7.05 (m, 5H); 6.6 (d, 1H,J=6.49); 5.24 (d, 1H); 5.14 (s, 2H); 4.93 (d, 1H, J=9.32); 4.0 (m, 2H);3.8 (m, 2H); 3.67 (m, 1H); 3.51 (d, 1H, J=7.46), 3.3-3.2 (m, 2H);3.0-2.84 (m; 2H); 2.18 (sep, 1H, J=7.46), 1.0 (m, 6H)

The invention claimed is:
 1. A compound having the formula (II)

or pharmaceutically acceptable salts and stereoisomers thereof.